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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K14/475—Growth factors; Growth regulators
  • C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
• • A—HUMAN NECESSITIES
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  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
  • A61K2039/5152—Tumor cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• the invention described herein relates to analogues of the long pentraxin PTX3 (PTX3) and their use for the preparation of a vaccine for the treatment of tumours.
• tumour in fact, is capable of concealing itself from host's immune system through reduced expression of its own antigens or through the ineffective presentation of said antigens. It is known that both the class I major histocompatibility complex (MHC I) and molecules with co- stimulatory activity such as CD80 and CD86 are poorly or not all expressed by tumour cells.
• MHC I major histocompatibility complex
• CD80 and CD86 are poorly or not all expressed by tumour cells.
• the tumour moreover, is capable of secreting cytokines with an immunosuppressive activity such as IL-10 and TGF ⁇ , the function of which is to de-energise lymphocytes activated against associated tumour antigens. On the whole, the tumour induces a state of immunological tolerance in the host.
• tumour cells modified, for example, by cytokines, by co-stimulatory molecules, bacteria or toxins, for the purposes of modifying the tumour cells and making them recognisable or capable of being processed by the immune system.
• PTX3 is a protein which is expressed in various cell types (Bottazzi et al., J. Biol Chem; 272: 32817-32823, 1997) particularly in mononuclear phagocytes and endothelial cells, after exposure to the inflammatory cytokines, Interleukin 1 beta (IL-1 beta) and Tumour Necrosis Factor alpha (TNF-alpha).
• IL-1 beta Interleukin 1 beta
• TNF-alpha Tumour Necrosis Factor alpha
• This protein consists of two structural domains, an N-terminal unrelated to any known molecule, and a C-terminal similar to the short pentraxins such as C-reactive protein (CRP).
• CRP C-reactive protein
• the PTX3 gene is located on mouse chromosome 3, in a region similar to the human region 3q (q24-28), in agreement with the documented location of hPTX3 in the region 3q 25.
• mouse PTX3 (mPTX3) (Introna M. et al., Blood 87 (1996, 1862- 1872) is very similar to hPTX3 on the basis of its organisation, location and sequence (Breviario F. et al., J. Biol. Chem. 267:22190, 1992).
• the degree of identity between the sequences is 82% between the human gene and the mouse gene, and as much as 92% if the conservative substitutions are considered.
• a further object of the invention described herein is a derivative of murine PTX3 with amino-acid sequence Seq. Id. No. 2.
• a further object of the invention described herein is a derivative of human PTX3 with amino-acid sequence Seq. Id. No. 3.
• a further object of the invention described herein is a derivative of human PTX3 with amino-acid sequence Seq. Id. No. 4.
• a further object of the invention described herein is a derivative of murine PTX3 biotinylated at random, with 1-100 molecules of biotin per single protein of PTX3, with amino-acid sequence Seq. Id. No. 5.
• a further object of the invention described herein is a derivative of human PTX3 biotinylated at random, with 1-100 molecules of biotin per single protein of PTX3, with amino-acid sequence Seq. Id. No. 6.
• a further object of the invention described herein is a Murine PTX3 cDNA having sequence Seq. Id. No. 7.
• a further object of the invention described herein is a Murine PTX3 cDNA having sequence Seq. Id. No. 8.
• a further object of the invention described herein is an autologous vaccine containing inactivated tumour cells of a solid or haematological tumour, bearing on their surface a derivative of PTX3 with amino-acid sequence Seq. Id. No. 1-6, and possibly an adjuvant.
• a further object of the invention described herein is a procedure for preparing an autologous vaccine, consisting of the following stages:
• tumour cells by means of known methods, from a patient suffering from a solid or haematological tumours;
• a further object of the invention described herein is a process for preparing an autologous vaccine consisting of the following stages:
• tumour cells - taking tumour cells from a patient suffering from a solid or haematological tumour; - inactivation, in vitro, of the tumour cells by means of known methods, e.g. radiation, in order to inhibit their proliferative ability;
• a further object of the invention described herein is the use of a vaccine prepared with the procedures outlined above for the preparation of a medicine which can be administered, for instance, by the subcutaneous, intravenous or intra-lymph-nodal routes for the treatment of tumours.
• a further object of the invention described herein is the use of a derivative of PTX3 with amino-acid sequence Seq. Id. No. 1-6, bound to the surface of the inactivated tumour cells of a solid or haematological tumour, for the preparation of an autologous vaccine which can be administered by the subcutaneous, intravenous or intra-lymph-nodal or other routes for the treatment of tumours.
• a further object of the invention described herein is the use of a vaccine, prepared with a derivative of PTX3 with amino-acid sequence Seq. Id. No.
• tumours in which said derivative is bound to the surface of the inactivated tumour cells of a solid tumour, for the preparation of a medicine which can be administered by the subcutaneous, intravenous, intra-lymph-nodal or other routes for the treatment of tumours.
• the tumour vaccine according to the invention described herein may contain one or more adjuvants that induce a non-specific immune response.
• adjuvants are Freund's complete adjuvant, Freund's incomplete adjuvant, bacterial preparations such as, for example, BCG, preparations of bacterial components such as tuberculin, naturally- occurring macromolecular substances such as mannan yeast, alum, synthetic adjuvants such as "Titer Max Gold” and the like.
• Other adjuvants can obviously also be used.
• the vaccine according to the invention can be inoculated in either the presence or absence of the adjuvant.
• Murine PTX3 cDNA (Introna M. et al, Blood 87 (1996) 1862-1872) was modified by the introduction of a sequence of 18 nucleotides coding for
• Murine PTX3 cDNA thus modified was cloned in the plasmid expression vector pcDNA 3.1 (Invitrogen) using the EcoRI and Xbal restriction sites (Ausubel F.M. et al, 1987, Current Protocols in Molecular
• This plasmid vector was called pPTX3/hisl.
• the plasmid vector was called ⁇ PTX3/his2.
• the plasmid vectors pPTX3/hisl and pPTX3/his2 were used for the transfection of COS7 cells with lipofectamine 2000 (Invitrogen) (Ciccarone et al, 1999 FOCUS 21, 54). After transfection with one of the two plasmids, these cells release an amino-acid sequence of the murine recombinant PTX3 into the culture medium (DMEM GIBCO) (the plasmid vector pPTX3/hisl codes for Seq. Id. No. 1, while plasmid vector p PTX3/his2 codes for Seq. Id. No. 2) recognised both by anti-PTX3 antibodies and by anti-histidine antibodies (Quiagen) ( Figure 2).
• PTX3hisl and PTX3his2 were purified by affinity chromatography, using Amersham Pharmacia Biotech columns (Histrap Kit). The passage of the dialysed supernatant of COS-7 cells transfected with one of the two vectors and the subsequent elution of the protein with a discontinuous gradient of imidazole from these columns, permits the recovery of approximately 60-80% of the recombinant PTX3 produced.
• the protein PTX3hisl shows an ability to decamerise (Figure 3a) and bind Clq ( Figure 3b) in a similar way to that described for the naturally occurring protein of PTX3.
• Murine PTX3 cDNA (Introna M. et al, Blood 87 (1996) 1862-1872) was subcloned in the expression vector pcDNA 3.1 (Invitrogen) e subsequently transfected in COS7 cells using lipofectamine 2000 (Invitrogen) (Ciccarone et al, 1999 FOCUS 21, 54).
• the recombinant protein thus obtained was purified from the culture supernatant of the COS7 cells by means of affinity chromatography, using an anti-PTX3 monoclonal antibody conjugated to protein G, with the procedure described by Bottazzi et al, J. Biol. Chem. 272(52):32817-32823, 1997.
• Biotin is a 244-dalton molecule capable of binding avidin and streptoavidin molecules with high affinity. Biotin was bound to amino-acid residues of human and mouse PTX3, or proteins of cell membranes of inactivated tumour cells using the chemical derivative NHS-LC-Biotin (PIERCE) (Altin et al, Anal Biochem 224: 382-389, 1995). The binding of biotin molecules both to the membranes of tumour cells and to recombinant PTX3 protein makes it possible to anchor PTX3 to the tumour cell.
• the molecules of avidin added to the mixture of PTX3 and tumour cells act as a molecular bridge between the biotins present on the cell membrane and those bound to the PTX3 amino acids.
• the lipid chelating agent NTA-DOGS (Avanti Polar Lipids Inc.) was prepared as a liposomal supension with liposomes with a mean diameter of approximately 500 nm.
• NTA-DOGS is intercalated in the lipid bilayer via its hydrophobic portion and exposes, on the cell surface, the polar head of nitrolotriacetic acid capable of binding any peptide or protein containing 6 histidine domains (Broekhoven et al. 2000 J. Immunology 164: 2433-2443).
• the efficiency of incorporation of the lipid chelating agent in the bilayer of the membrane of the P815 tumour cell line was measured using a 6-histidine peptide conjugated to a biotin molecule.
• FACS fluorescence activated cell sorter
• analysis of the P815 cells treated with liposomes of NTA-DOGS (P815-NTA), with the biotinylated peptide and lastly with fluorescinated streptoavidin revealed an approximately 100-fold increase in the fluorescent signal compared to controls (P815 treated with the biotinylated peptide alone).
• the protein PTX3/hisl is capable of binding to the membrane surface of tumour cells treated with liposomes of the lipid chelating agent NTA-DOGS.
• the protein PTX3/his 1 purified from the supernatant of COS-7 cells and incubated with P815-NTA cells is capable of binding to their membrane surface.
• FACS analysis of P815-NTA cells using anti-PTX3 antibodies revealed a 10-fold greater fluorescent signal than P815 controls not treated with the recombinant protein ( Figure 4). This result confirms that binding of PTX3/hisl to the P815 cell membrane has taken place.
• tumour cells 1, 2, 3 or 4, bound to tumour cells
• tumour cells (10-100 million) are taken, by means of known methods, from a patient suffering from a solid tumour.
• tumour cells are treated with liposomes of the lipid chelating agent NTA-DOGS (50-250 ⁇ M).
• tumour cells are further treated with a derivative of PTX3 (50-500 ⁇ g/ml) with amino-acid sequence Seq. Id. No. 1, 2, 3 or 4, in order to bind said derivative of PTX3 to the membranes of said tumour cells.
• a derivative of PTX3 50-500 ⁇ g/ml
• amino-acid sequence Seq. Id. No. 1, 2, 3 or 4 in order to bind said derivative of PTX3 to the membranes of said tumour cells.
• An aliquot of tumour cells thus modified is subjected to
• modified tumour cells with the PTX3 derivative bound to the membranes, are inoculated into the patient from whom they have come (autologous vaccine) by means of administration via the subcutaneous, intravenous, intra-lymph- nodal or other routes.
• autologous vaccine an autologous vaccine
• tumour cells (10-100 million) are taken, by means of known methods, from a patient suffering from a tumour.
• tumour cells are inactivated, by means of known methods, in vitro, in order to inhibit their proliferative activity, for example, by radiation.
• the inactivated tumour cells are subjected to biotinylation (100-1000 biotins/cell).
• biotinylated tumour cells are incubated with avidin (10-100 ⁇ g/ml).
• tumour cells To the cell membrane of the tumour cells incubated with avidin (as in para, "d") is bound a biotinylated PTX3 derivative (50- 500 ⁇ g/ml) with amino-acid sequence Seq. Id. No. 5 or 6.
• a biotinylated PTX3 derivative 50- 500 ⁇ g/ml with amino-acid sequence Seq. Id. No. 5 or 6.
• the modified tumour cells with the PTX3 bound to the membranes (as in para, "e") are inoculated into the patient from whom they have come (autologous vaccine) by means of administration via the subcutaneous, intravenous, intra-lymph- nodal or other routes.
• DBA2J mice were inoculated subcutaneously with lxl 0 5 murine P815 tumour cells.
• a third group of animals (n 10) was treated with parental P815 cells (P815).
• tumour sizes were measured in the three weeks following inoculation of the cells on the days indicated in the table, by direct measurement with a Vernier calliper. The calculation of tumour size in mm 3 was done using the formula [(width 2 x length)/ 2].

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